The confinement of single molecules, e.g. DNA, within nanoscale environments is crucial within a range of research fields including, but not limited to, biomedical research, enhanced genetic diagnosis and physical studies. For example, the direct visualization of an individual stretched DNA molecule allows the acquisition of contextual information along the DNA molecule. It also allow for organisms, in particular microorganisms responsible for disease to be identified without requiring steps such as sample culturing, DNA amplification etc. which today form bottlenecks within prior art diagnostic methodologies.
Single molecule confinement and nanoscale environment manipulation of molecules when coupled to the advancements of technology in nanofabrication offers the potential for high throughput nano-molecular devices for molecular research and development, diagnosis, etc. Nanoscale confinement (nano-confinement) based manipulation of molecules when compared to the prior art single molecule manipulation technique such as tweezer technology and surface/hydrodynamic stretching offers several advantages. First, nanofabrication technologies allow highly parallel devices through integration providing high throughput analysis. Second, they can be easily integrated with nano- and micro-fluidic elements for cycling molecules and allowing upstream/downstream pre- and post-processing.
Within the prior art techniques exploiting nano-fluidics devices single DNA molecules are confined and extended along the nano-channels through the establishment of a pressure gradient along the nano-channel. Depending upon the dimensions of the nano-channel the molecules conformation is molded by the surrounding geometry from a three-dimensional (3D) coil shape to a one-dimensional (1D) extended conformation. However, high hydraulic resistance of the confinement area and free energy barrier at the edge of the nano-channels lead to limited fluid transport and practical nano-channel dimensions. Further, in conventional nano-fluidic technology, high hydrodynamic forces are required to drive the molecules into the nano-channels, potentially leading to fragmentation of large molecules. One prior art approach to overcome these nano-fluidic technology drawbacks is that of Convex-Lens Induced Confinement (CLIC) or Convex Lens-Induced Nanoscale Templating (CLINT) that traps molecules between a nano-patterned substrate and a convex surface. However, CLIC/CLINT limits both buffer exchange for subsequent processes and the concentration of confined molecules within a single field of view. Moreover, confinement varies rapidly above the nano-patterned area from the convex upper surface, limiting the size of the confinement area and the accessibility of the whole device.
Accordingly, it would be beneficial to provide a new technology option that leverages the benefits of nano-scale confinement and nanoscale manufacturing methodologies to provide a means to confine large numbers of molecules within a single field of view. It would be further beneficial to provide a technology allowing for uniform trapping-confinement, extension, and optical observation of single molecules within open and uniform environment without requiring hydrodynamic force, mechanical components or the need for very thin (nanoscale) vertical device dimensions. Further, it would be beneficial for this technology to exploit high volume, low cost automated manufacturing methodologies as well as providing compatibility with nano-fluidic and micro-fluidic technologies for automated processing of samples.